The thymus is the primary site of T cell maturation, and as such, it plays a major role in the establishment and maintenance of the peripheral T cell pool throughout the lifespan of the animal. Measuring thymic output without prior experimental manipulation is key to determining the age-dependence of the quantity and quality of cells exported by the thymus. A novel system has been devised for marking recent thymic emigrants (RTEs), using mice transgenic for green fluorescent protein (GFP) under the control of the RAG2 promoter. While thymocytes from these mice begin expressing GFP at the expected developmental stage, GFP lingers in these cells after RAG2 expression is extinguished. The resulting population of GFP(hi) peripheral T cells are RTEs because they disappear within one week of thymectomy. Preliminary analyses of GFP(hi) peripheral T cells from RAG2pGFP transgenic mice indicate that RTEs undergo a phenotypic and functional maturation in the weeks after they reach the lymphoid periphery. Within this population of new peripheral immigrants, the CD4:CD8 ratio is higher, CD24 expression is higher, and Qa-2 expression is lower than found on more mature peripheral T cells. CD8+ RTEs contain approximately half the expected cytolytic precursors, and without exogenous IL-2, CD4+ RTEs proliferate poorly upon T cell receptor crosslinking. One aim of the current proposal is to complete this functional and phenotypic characterization of RTEs from unmanipulated young adult mice and to extend these analyses to younger and older individuals. Age-dependent quantitation of murine RTEs and characterization of their function and surface antigen phenotype will help determine the extent of emigration from the involuting organ. A second aim is to investigate the means by which the CD4:CD8 ratio declines as the RTE population becomes incorporated into the pool of mature peripheral T cells in the young adult mouse. Whether this adjustment is driven by the loss of CD4+ RTEs, preferential proliferation of CD8+ RTEs, or some combination of these factors, the age-dependence of this adjustment will be analyzed. The final aim is to analyze the functional heterogeneity of RTEs in mice of various ages to determine the composition of the RTE compartment and to measure the antigen receptor repertoire. The overall goal of this work is to understand how thymic output and post thymic maturation vary with age, with an eye toward modulating immune senescence and recovery from lymphoablative therapies and diseases.